EP2834351B1 - Metabolitaktivität mit veränderten enzymen - Google Patents

Metabolitaktivität mit veränderten enzymen Download PDF

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EP2834351B1
EP2834351B1 EP13772074.4A EP13772074A EP2834351B1 EP 2834351 B1 EP2834351 B1 EP 2834351B1 EP 13772074 A EP13772074 A EP 13772074A EP 2834351 B1 EP2834351 B1 EP 2834351B1
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clostridium
substrate
alcohol dehydrogenase
seq
mutant
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French (fr)
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EP2834351A1 (de
EP2834351A4 (de
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Michael Koepke
Wayne Michael Patrick
Danielle Joan MADDOCK
Monica GERTH
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Lanzatech NZ Inc
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Lanzatech New Zealand Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C12P7/28—Acetone-containing products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01002—Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to mutant alcohol dehydrogenase enzymes, microorganisms comprising same, and methods for the production of one or more products by microbial fermentation.
  • Butanol is usually used as solvent or biofuel, while acetone is considered as an unwanted by-product [ Duerre P: Production of solvents. In: Handbook on Clostridia, CRC Press, 2005: 671-696 ].
  • a few isolates of Clostridium beijerinckii are known to produce isopropanol instead of acetone due to a secondary alcohol dehydrogenase [ George HA, Johnson JL, Moore WEC, Holdeman LV, Chen JS: Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum.
  • Isopropanol has similar properties as butanol and would be beneficial over acetone. However, the reduction is not very efficient and respective Clostridium beijerinckii strains don't produce good titers and are not considered as useful production strains.
  • C . acetobutylicum has been metabolically engineered for isopropanol production using a secondary alcohol dehydrogenase from C . beijerinckii [ Lee et al, 2012: Metabolic engineering of Clostridium acetobutylicum ATCC824 for isopropanol-butanol-ethanol fermentation, Appl. Environ. Microbiol. 78: 1416-1423 ], but a highly efficient alcohol dehydrogenase would be required to optimize this process.
  • a challenge of the ABE fermentation is that all known organisms rely on sugar or starch based substrates.
  • the cost of many carbohydrate feed stocks suitable for the production of chemical products such as acetone and isoproanol is influenced by their value as human food or animal feed, and the cultivation of starch or sucrose-producing crops for such production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower cost and/or more abundant carbon resources into useful chemical products such as acetone and isopropanol.
  • CO is a major free energy-rich by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products.
  • organic materials such as coal or oil and oil derived products.
  • the steel industry in Australia is reported to produce and release into the atmosphere over 500,000 tonnes of CO annually.
  • Acetogenic organisms such as the closely related microorganisms Clostridium autoethanogenum, C. ljungdahlii , and C.
  • ragsdalei are able to grow chemoautotrophically on CO or CO 2 /H 2 containing gases as sole energy and carbon source and synthesize products such as acetate, ethanol, or 2,3-butanediol, but neither acetone nor isopropanol [ Munasinghe PC, Khanal SK: Biomass-derived syngas fermentation into biofuels: Opportunities and challenges. Bioresource Technol 2010, 5013-22 ].
  • Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity that unlike the linker primary alcohol dehydrogenase, the Rossmann-fold-containing Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase binds its catalytic zinc atom using a sorbitol dehydrogenase-like Cys-His-Asp motif and does not bind a structural zinc atom [ Biochem J 1997, 326:717-724 ].
  • the inventors have identified a novel primary: secondary alcohol dehydrogenase in C . autoethanogenum which could be used for the production of isopropanol and/or one or more other products from CO or to upgrade acetone-butanol-ethanol (ABE) fermentation to isopropanol-butanol-ethanol (IBE) fermentation or for the conversion of MEK to 2-butanol, for example, and have optimized the properties of the enzyme, such as substrate and/or co-factor specificity, by directed mutagenesis.
  • ABE acetone-butanol-ethanol
  • IBE isopropanol-butanol-ethanol
  • an alcohol dehydrogenase selected from SEQ ID NO: 42, SEQ ID NO: 48, or SEQ ID NO: 50.
  • a nucleic acid encoding an alcohol dehydrogenase of the first aspect.
  • nucleic acid of the second aspect wherein the nucleic acid comprises SEQ ID NO: 41, SEQ ID NO: 47, or SEQ ID NO: 49.
  • nucleic acid vector comprising the nucleic acid of the second aspect.
  • a recombinant microorganism comprising a nucleic acid of the third aspect.
  • a recombinant microorganism of the fifth aspect wherein the recombinant microorganism is capable of producing one or more of isopropanol, ethanol, 2,3-butanediol, 2-butanol, acetoin, MEK, acetaldehyde, and acetone, and optionally one or more other products.
  • a recombinant microorganism of the fifth aspect wherein the recombinant microorganism is selected from the group consisting of bacteria, archaea, and fungi.
  • a recombinant microorganism of the fifth aspect wherein the recombinant microorganism is a carboxythrophic acetogen selected from the group consisting of Acetobacterium woodii , Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Moorella thermoacetica, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium coskatii, Clostridium aceticum, Clostridium magnum, Clostridium sp., Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigi
  • a recombinant microorganism of the fifth aspect wherein the recombinant microorganism is an ABE fermenting microorganism selected from the group comprising Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum.
  • a recombinant microorganism of the fifth aspect wherein the recombinant microorganism is selected from the group comprising Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Bacillus licheniformis, Bacillus subtilis, Lactobacillus brevis, and Saccharomyces cerevisiae.
  • an eleventh aspect of the present invention there is provided a method for the production of one or more of isopropanol, ethanol, 2,3-butanediol, 2-butanol, acetoin, MEK, acetaldehyde, and acetone, and optionally one or more other products, by microbial fermentation of a substrate using a microorganism as claimed in any one of the fifth to the tenth aspects.
  • the substrate is chosen from a substrate comprising one or more of CO, CO2, and H2 and/or a substrate comprising one or more carbohydrate.
  • the substrate comprises a CO-containing gas produced by an industrial process.
  • a fourteenth aspect of the present invention there is provided a method of the thirteenth aspect, wherein the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, and coke manufacturing.
  • the disclosure may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates.
  • the inventor(s) have unexpectedly found that mutation of an alcohol dehydrogenase enzyme from a carboxydotrophic acetogenic microorganism increases its specificity for one or more of the substrates acetoin, methyl ethyl ketone (MEK or 2-butanone), acetone and acetalydehyde relative to one another.
  • MEK methyl ethyl ketone
  • mutants that are able to accept NADH as a co-factor instead of or in addition to NADPH.
  • NADH as a co-factor instead of or in addition to NADPH.
  • the mutation(s) mean that enzymes of the invention are adapted to preferentially produce one fermentation end product over another and allow one to utilize the typically much bigger NADH pool over the NADPH pool (Bennett & San, 2009).
  • ABE fermentation both ethanol and acetone are produced and in IBE fermentation isopropanol is produced (Köpke & Dürre, 2011).
  • the invention may allow one to increase the reduction of acetone to isopropanol or preferentially catalyse this reaction over acetaldehyde to ethanol or vice versa.
  • the invention may help to overcome limitations in isopropanol production, as all strains rely on an unmodified alcohol dehydrogenase that is strictly NADPH dependent and has a high background activity towards acetaldehyde.
  • E. coli strains engineered for isopropanol production suffer the same shortcomings and low yields as the same NADPH dependent alcohol dehydrogenase enzyme from C . beijerinckii is used without alternatives [ Hanai T et al (2007) Engineered synthetic pathway for isopropanol production in Escherichia coli.
  • the invention provides a means for the production of isopropanol from substrates comprising carbon monoxide by carboxydotrophic acetogenic microorganisms previously not able to produce viable levels of isopropanol. Some carboxydotrophic organisms as C .
  • autoethanogenum C. ljungdahlii or C . ragsdalei are able to form both ethanol and 2,3-butanediol (Köpke et al., 2011).
  • the invention may also allow one to increase the reduction of acetoin to 2,3-butanediol or preferentially catalyse this reaction over acetaldehyde to ethanol or vice versa.
  • 2,3-butanediol can be converted to MEK by a diol dehydratase ( Toraya et al, 1976, Substrate specificity of coenzyme B12-dependent diol dehydrase - glycerol as both a good substrate and a potent inactivator. Biochem. Biophys. Res. Commun., 69: 475-80 ).
  • the invention allows effective conversion of MEK into 2-butanol. Accordingly, the invention also provides a solution for producing 2,3-butanediol from acetoin and 2-butanol from MEK with higher specificity.
  • the invention provides, inter alia, alcohol dehydrogenases having an increased specificity for an acetone substrate over other substrates like acetoin, methyl ethyl ketone (MEK or 2-butanone) and/or an acetaldehyde substrate, an MEK substrate over an acetaldehyde and/or acetoin substrate, an acetaldehyde substrate over an MEK, acetoin and/or acetone substrate, and/or an acetoin substrate over an acetone, MEK and/or acetaldehyde substrate wherein the alcohol dehydrogenase includes at least one or more mutation compared to the corresponding wild type alcohol dehydrogenase, nucleic acids encoding the alcohol dehydrogenases, nucleic acid vectors comprising the nucleic acids, microorganisms which are capable of producing one or more of isopropanol, 2,3-butanediol, ethanol and 2-butanol, and optionally one or more
  • the invention also provides an alcohol dehydrogenase that has substantially no ability to use acetoin as a substrate, nucleic acids and nucleic acid vectors encoding such an alcohol dehydrogenase, microorganisms comprising said nucleic acids or nucleic acid vectors and methods of use of such alcohol dehydrogenase.
  • substrate comprising one or more of CO, CO 2 and H 2 should be understood to include any substrate in which one or more of CO, CO 2 and H 2 is available to one or more strains of microorganisms for growth and/or fermentation, for example. It should be appreciated that the substrate may comprise 100% CO, CO 2 or H 2 or a majority of CO, CO 2 or H 2 compared to the other gases, or may be combined in any ratio of two or more of the gases. In particular embodiments, the substrate comprises a combination of CO and CO 2 . In another embodiment the substrate comprises a combination of CO and H 2 . In another embodiment, the substrate comprises a combination of CO 2 and H 2 . In another embodiment, the substrate comprises a combination of CO, CO 2 and H 2 .
  • the substrate may comprise CO 2 and any culture, growth or fermentation performed in the presence of light (photosynthesis) and/or electricity (electrosynthesis).
  • the CO 2 is combined with O 2 .
  • the "substrate comprising CO, CO 2 and H 2 " is a “substrate comprising carbon monoxide”.
  • a “substrate comprising CO” and like terms should be understood to include any substrate in which carbon monoxide is available to one or more strains of microorganisms for growth and/or fermentation, for example.
  • gaseous substrate comprising carbon monoxide includes any gas which contains a level of carbon monoxide.
  • the substrate contains at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume.
  • the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
  • the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H 2 :CO.
  • the substrate comprises about 30% or less H 2 by volume, 20% or less H 2 by volume, about 15% or less H 2 by volume or about 10% or less H 2 by volume.
  • the substrate stream comprises low concentrations of H 2 , for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free.
  • the substrate may also contain some CO 2 for example, such as about 1% to about 80% CO 2 by volume, or 1% to about 30% CO 2 by volume. In one disclosure the substrate comprises less than or equal to about 20% CO 2 by volume. In particular disclosures the substrate comprises less than or equal to about 15% CO 2 by volume, less than or equal to about 10% CO 2 by volume, less than or equal to about 5% CO 2 by volume or substantially no CO 2 .
  • the CO-containing gaseous substrate is an industrial off or waste gas.
  • “Industrial waste or off gases” should be taken broadly to include any gases comprising CO produced by an industrial process and include gases produced as a result of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, and coke manufacturing. Further examples may be provided elsewhere herein.
  • the "substrate comprising CO, CO 2 and H 2 " is a “substrate comprising CO 2 and H 2 ".
  • a “substrate comprising CO 2 and H 2 " and like terms should be understood to include any substrate in which carbon dioxide and hydrogen is available to one or more strains of microorganisms for growth and/or fermentation, for example.
  • the CO 2 and H 2 containing substrate will typically contain a major proportion of H 2 , such as at least about 30% H 2 by volume, or at least 40% H 2 by volume, or at least 50% H 2 by volume, or at least 60% H 2 by volume, or at least 70% H 2 by volume, or at least 80% H 2 by volume, or at least 85% H 2 by volume.
  • the gaseous substrate will typically contain at least about 10% CO 2 by volume, or at least 15% CO 2 by volume, or at least 20%CO 2 by volume, or at least 25% CO 2 by volume, or at least 30% CO 2 by volume, or at least 40% CO 2 by volume.
  • the ratio of H2: CO2 is around 1:1, or around 2:1, or around 3:1.
  • the substrate comprising CO 2 and H 2 is a waste gas obtained as a by-product of an industrial process, or from some other source.
  • the largest source of CO 2 emissions globally is from the combustion of fossil fuels such as coal, oil and gas in power plants, industrial facilities and other sources.
  • the gaseous substrate may be a CO 2 and H 2 -containing waste gas obtained as a by-product of an industrial process, or from some another source such as from automobile exhaust fumes.
  • the industrial process is selected from the group consisting of hydrogen manufacture, ammonia manufacture, combustion of fuels, gasification of coal, and the production of limestone and cement.
  • the gaseous substrate may be the result of blending one or more gaseous substrates to provide a blended stream. It would be understood to a skilled person that waste gas streams rich in H 2 or rich in CO 2 are more abundant that waste gas streams rich in both H 2 and CO 2 . A skilled person would understand that one or more gas streams comprising one of the desired components of CO 2 and H 2 could be blended.
  • Hydrogen rich gas streams are produced by a variety of processes including steam reformation of hydrocarbons, and in particular steam reformation of natural gas.
  • the partial oxidation of coal or hydrocarbons is also a source of hydrogen rich gas.
  • Other sources of hydrogen rich gas include the electrolysis of water, by-products from electrolytic cells used to produce chlorine and from various refinery and chemical streams.
  • Gas streams typically rich in Carbon dioxide include exhaust gasses from combustion of a hydrocarbon, such as natural gas or oil. Carbon dioxide is also produced as a by-product from the production of ammonia, lime or phosphate.
  • gaseous substrate containing CO or a “gaseous substrate comprising one or more of CO, CO 2 and H 2 ".
  • the gaseous substrate may be provided in alternative forms.
  • the gaseous substrate may be provided dissolved in a liquid.
  • a liquid is saturated with a carbon monoxide, carbon dioxide and/or hydrogen containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology.
  • a microbubble dispersion generator Hensirisak et. al.
  • the gaseous substrate containing CO may be adsorbed onto a solid support.
  • substrate containing CO “substrate comprising CO 2 and H 2 ) and “substrate comprising one or more of CO, CO 2 and H 2 " and like phrases.
  • substrate comprising one or more carbohydrates should be understood to include any substrate in which one or more carbohydrate is available to one or more strains of microorganisms for growth and/or fermentation, for example.
  • Carbohydrates should be taken broadly to include mono-, di-, oligo- and poly-saccharides, simple and complex carbohydrates, including glucose, fructose, molasses and starch.
  • the "substrate comprising one or more carbohydrates” may be sourced from biomass.
  • the biomass may be of any nature and includes, for example, residues from forests or other commercial crops (such as trees, branches, stumps, wood chips, saw dust, clippings), municipal solid waste, and crops grown to provide a feedstock for the production of one or more products by microbial fermentation, including, for example, miscanthus, switchgrass, hemp, corn, poplar, willow, sorghum, sugarcane, and bamboo.
  • the phrases "fermenting”, “fermentation process” or “fermentation reaction” and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process.
  • the bioreactor may comprise a first growth reactor and a second fermentation reactor.
  • the addition of metals or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.
  • bioreactor includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.
  • CSTR Continuous Stirred Tank Reactor
  • ICR Immobilized Cell Reactor
  • TBR Trickle Bed Reactor
  • Bubble Column Gas Lift Fermenter
  • Static Mixer Static Mixer
  • nucleic acid “constructs” or “vectors” and like terms should be taken broadly to include any nucleic acid (including DNA, cDNA and RNA) suitable for use as a vehicle to transfer genetic material into a cell.
  • the terms should be taken to include plasmids, viruses (including bacteriophage), cosmids and artificial chromosomes.
  • Constructs or vectors may include one or more regulatory elements, an origin of replication, a multicloning site and/or a selectable marker.
  • the constructs or vectors are adapted to allow expression of one or more genes encoded by the construct or vector.
  • Nucleic acid constructs or vectors include naked nucleic acids as well as nucleic acids formulated with one or more agents to facilitate delivery to a cell (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).
  • the vectors may be used for cloning or expression of nucleic acids and for transformation of microorganisms to produce recombinant microorganisms.
  • the vectors may include one or more nucleic acids encoding one or more alcohol dehydrogenase enzyme of the invention.
  • Exogenous nucleic acids are nucleic acids which originate outside of the microorganism to which they are introduced. Exogenous nucleic acids may be derived from any appropriate source, including, but not limited to, the microorganism to which they are to be introduced, strains or species of microorganisms which differ from the organism to which they are to be introduced, or they may be created artificially or by recombination. In another disclosure, the exogenous nucleic acids represent nucleic acid sequences not naturally present within the microorganism to which they are to be introduced and allow for the expression of a product not naturally present within the microorganism. The exogenous nucleic acid may be adapted to integrate into the genome of the microorganism to which it is to be introduced or to remain in an extra-chromosomal state.
  • a "parental microorganism” is a microorganism used to generate a microorganism of the invention.
  • the parental microorganism may be one that occurs in nature (ie a wild type microorganism) or one that has been previously modified but which does not express or over-express one or more of the enzymes the subject of the present invention. Accordingly, the microorganisms of the invention have been modified to express one or more alcohol dehydrogenase of the invention in the parental microorganism.
  • the alcohol dehydrogenase enzymes of the invention are referred to herein to have "increased specificity" for one substrate over another. This is intended to mean that the alcohol dehydrogenase has increased specificity for one substrate relative to another, compared to the wild type alcohol dehydrogenase. It should not be taken to necessarily infer that an alcohol dehydrogenase of the invention has a higher specificity for a particular substrate compared to the wild type alcohol dehyrodenase, although this may be the case. Additionally, the term should not be taken to mean that an alcohol dehydrogenase of the invention has absolute specificity for a particular substrate over another, although this may be the case, and includes at least a preference for a particular substrate over another substrate.
  • “Increased specificity”, “higher specificity” or like terms when used in relation to an NADH or NADPH co-factor, refers to the degree of affinity with which a co-factor binds to an alcohol dehydrogenase during a reaction. It should not be taken to mean that an alcohol dehydrogenase and a co-factor have absolute specificity, although this may be the case, and includes at least a preference for the binding between a particular alcohol dehydrogenase and one co-factor over another co-factor.
  • an alcohol dehydrogenase of the invention has substantially no ability to use acetoin as a substrate and so acetoin may accumulate.
  • one or more of the products referred to herein including one or more of isopropanol, 2,3-butanediol, ethanol and 2-butanol, may be used as intermediates or precursors which are further converted into downstream products, in the same fermentation reaction, a separate fermentation reaction, or by chemical synthesis.
  • one may not be able to detect the production of one or more of the products in a particular microorganism or may only be able to detect small levels of production.
  • the production of the one or more products may be inferred based on the production of one or more downstream product.
  • an alcohol dehydrogenase of the invention has "substantially no ability to use acetoin as a substrate". This does not necessarily imply that the enzyme has absolutely no ability to use acetoin as a substrate, although this may be preferred.
  • the phrase should be taken to include a tolerance of approximately 1% or less of the activity of a wild type enzyme or an enzyme efficiency of kcat/K M of less than 0.1 sec -1 mM -1 .
  • the group of alcohol dehydrogenases will have at least approximately 65% sequence identity to the alcohol dehydrogenase of SEQ ID 36, more particularly at least approximately 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the invention may be applied to the following alcohol dehydrogenases: primary: secondary alcohol deheydrogenase of C . autoethanogenum (SEQ ID No 36), primary: secondary alcohol deheydrogenase of C . ljungdahlii (YP_003780646.1), primary: secondary alcohol deheydrogenase of C . beijerinckii (P25984.2), primary: secondary alcohol deheydrogenase of Thermoanaerobacter ethanolicus (ABC50090.1), or primary: secondary alcohol deheydrogenase of Thermoanaerobium brockii (P14941.1).
  • An alcohol dehydrogenase of the invention comprises at least one mutation compared to the corresponding wild type alcohol dehydrogenase.
  • the at least one mutation may be an amino acid substitution at one or a combination of the amino acids corresponding to position Gly198, Ser199, Arg200, Pro201 and Tyr218 of the alcohol dehydrogenase sequence of SEQ ID 36.
  • the alcohol dehydrogenase comprises at least one mutation wherein the at least one mutation is an amino acid substitution at the position corresponding to position Ser199 of the alcohol dehydrogenase sequence of SEQ ID 36.
  • the alcohol dehydrogenase may include one or more of the following mutations compared to the corresponding wild type alcohol dehydrogenase: Gly198Asp, Gly198Ile, Gly198Leu, Gly198Val, Ser199Asp, Ser199Glu, Ser199Leu, Ser199Val, Arg200Glu, Pro201Asp, Pro201Glu, Tyr218Ala and Tyr218Phe.
  • the inventors also envisage alcohol dehydrogenases of the invention including one of the following mutations compared to the corresponding wild type alcohol dehydrogenase: Tyr218Gly, Tyr218Ser and Tyr218Val. These mutations represent substitutions which are all close in size to the Tyr218Ala and Tyr218Phe substitutions exemplified in the examples section herein after.
  • the alcohol dehydrogenase includes a Ser199Asp substitution. In one embodiment, the alcohol dehydrogenase includes a Ser199Glu substitution.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, Pro201Glu. In one disclosure, the alcohol dehydrogenase includes a combination of the following substitutions, Gly198Asp, Ser199Leu, and Pro201Glu. In another embodiment, the alcohol dehydrogenase includes a combination of the following substitutions: Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Ala. In another embodiment, the alcohol dehydrogenase includes a combination of the following substitutions: Gly198Asp, Ser199Va1, Pro201Glu, and Tyr218Phe.
  • the alcohol dehydrogenase includes a combination of the following substitutions: Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Gly. In another disclosure, the alcohol dehydrogenase includes a combination of the following substitutions: Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Ser. In another disclosure, the alcohol dehydrogenase includes a combination of the following substitutions: Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Val.
  • the alcohol dehydrogenase includes a Ser199Asp substitution and has an increased substrate specificity for 1) acetone over MEK and/or acetoin. In one embodiment, the alcohol dehydrogenase includes a Ser199Asp substitution and has an increased substrate specificity for acetone over MEK and acetoin.
  • the alcohol dehydrogenase includes a Ser199Glu substitution and has an increased substrate specificity for 1) acetone over MEK, acetaldehyde and/or acetoin; and/or 2) MEK over acetaldehyde and/or acetoin.
  • the alcohol dehydrogenase includes a Ser199Glu substitution and has an increased substrate specificity for 1) acetone over MEK and acetaldehyde and acetoin.
  • the alcohol dehydrogenase includes a Ser199Glu substitution and has an increased substrate specificity for MEK over acetaldehyde and acetoin.
  • the alcohol dehydrogenase includes a Ser199Glu substitution and has an increased substrate specificity for 1) acetone over MEK and acetaldehyde and acetoin; and 2) MEK over acetaldehyde and acetoin.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, and Pro201Glu and has an increased substrate specificity for 1) acetone over MEK; and/or 2) acetaldehyde over MEK, acetone and/or acetoin; and/or, 3) acetoin over acetone and/or MEK.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for acetaldehyde over MEK and acetone and acetoin.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for acetoin over acetone and MEK.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for 1) acetone over MEK; and 2) acetaldehyde over MEK and acetone and acetoin; and 3) acetoin over acetone and MEK.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Ala has an increased substrate specificity for 1) acetone over MEK, acetaldehyde and/or acetoin; and/or 2) MEK over acetaldehyde and/or acetoin; and/or 3) acetoin over acetaldehyde.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for acetone over MEK and acetaldehyde and acetoin.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for MEK over acetaldehyde and acetoin. In another embodiment, an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for 1) acetone over MEK and acetaldehyde and acetoin; and 2) MEK over acetaldehyde and acetoin; and 3) acetoin over acetaldehyde.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Phe and has an increased substrate specificity for 1) acetone over MEK, acetaldehyde and/or acetoin; and/or MEK over acetaldehyde and/or acetoin; and/or 3) acetalydehyde over acetoin.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for acetone over MEK and acetaldehyde and acetoin.
  • an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for MEK over acetaldehyde and acetoin. In another embodiment, an alcohol dehydrogenase including this combination of substitutions has an increased substrate specificity for 1) acetone over MEK, acetaldehyde and acetoin; and 2) MEK over acetaldehyde and acetoin; and 3) acetalydehyde over acetoin. In one embodiment, an alcohol dehydrogenase including this combination of substitutions has substantially no ability to use acetoin as a substrate.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Ala and is able to use NADH as a co-factor.
  • the alcohol dehydrogenase includes all of these substitutions and has an increased substrate specificity for 1) acetone over MEK, acetaldehyde and acetoin; and 2) MEK over acetaldehyde and acetoin; and 3) acetoin over acetaldehyde; and 4) is able to use NADH as a co-factor.
  • the alcohol dehydrogenase includes a combination of the following substitutions Gly198Asp, Ser199Val, Pro201Glu, and Tyr218Phe and is able to use NADH as a co-factor.
  • the alcohol dehydrogenase includes all of these substitutions and has an increased substrate specificity for for 1) acetone over MEK, acetaldehyde and acetoin; and 2) MEK over acetaldehyde and acetoin; and 3) acetalydehyde over acetoin; and 5) is able to use NADH as a co-factor.
  • the alcohol dehydrogenase has the sequence provided in SEQ ID 38. In one embodiment, the alcohol dehydrogenase has the sequence provided in SEQ ID 42. In one embodiment, the alcohol dehydrogenase has the sequence provided in SEQ ID 50.
  • the alcohol dehydrogenase has the sequence provided in SEQ ID 44. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 46. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 48. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 52. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 54. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 63. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 64. In one disclosure, the alcohol dehydrogenase has the sequence provided in SEQ ID 65.
  • the alcohol dehydrogenases of the invention may be made by any appropriate means known in the art including, for example, site directed mutagenesis techniques, random mutagenesis techniques, recombinant methodology and chemical synthesis, as described herein after.
  • mutant alcohol dehydrogenases of the invention may be naturally insoluble. These enzymes may be made soluble using standard techniques.
  • techniques involving, co-expression of one or more chaperones may be used.
  • co-expression of the GroEL and/or GroES chaperones is employed.
  • use of the plasmid pGro7 (Takara Bio, Inc; clontech.com/takara/NZ/Products/Protein_Research/Protein_Folding_and_Expression/Chaperone_Pla smid_Set) may be used.
  • This plasmid facilitates arabinose-inducible expression of the GroEL/ES chaperone proteins.
  • Exemplary techniques are also provided in Example 2 herein after.
  • an alcohol dehyrogenase of the invention may assess whether an alcohol dehyrogenase of the invention has the appropriate functionality using any number of known methods. However, by way of example, the methods outlined in the Examples herein after may be used. Alternatively, the methods outlined in Ismail et al. [Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. J Bacteriol 1993, 175: 5097-5105 ], or Khorkin et al [NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii. J Mol Biol. 1998, 22: 278(5): 967-981 ] may be used to assess enzyme activity.
  • Co-factor specificity may be assessed using standard methodology. However, by way of example the methods described in the "Examples" section herein after may be used.
  • the invention in so far as the invention relates to novel alcohol dehydrogenases, it also provides nucleic acids encoding the alcohol dehydrogenases and nucleic acid vectors comprising such nucleic acids.
  • nucleic acid encoding an alcohol dehydrogeanse of the invention, having regard to the amino acid sequence of the enzyme and the degeneracy in the genetic code.
  • the nucleic acid has the sequence of SEQ ID 37.
  • nucleic acid has the sequence of SEQ ID 41, SEQ ID NO: 47 or SEQ ID 49.
  • nucleic acid has the sequence of SEQ ID 39, SEQ ID 43, SEQ ID 45, SEQ ID 51, SEQ ID 53, SEQ ID 67, SEQ ID 68, SEQ ID 69 or SEQ ID 70.
  • nucleic acids encoding an alcohol dehydrogenase of the invention may be codon optimised for any particular microorganism.
  • nucleic acids, alcohol dehydrogenases and microorganisms of the invention may be made and used using recombinant technology
  • the invention also provides nucleic acid vectors comprising one or more nucleic acid encoding one or more alcohol dehydrogenase of the invention.
  • nucleic acids of the invention may remain extra-chromosomal upon transformation of a microorganism or may be adapted for integration into the genome of the microorganism. Accordingly, nucleic acids of the invention may include additional nucleotide sequences adapted to assist integration (for example, a region which allows for homologous recombination and targeted integration into the host genome) or stable expression and replication of an extrachromosomal construct (for example, origin of replication, promoter and other regulatory sequences).
  • the nucleic acids encoding one or more alcohol dehydrogenase of the invention may comprise a promoter adapted to promote expression of the one or more enzymes encoded by the nucleic acids.
  • the promoter is a constitutive promoter that is preferably highly active under appropriate fermentation conditions. Inducible promoters could also be used.
  • the promoter is selected from the group comprising Wood-Ljungdahl gene cluster or an arabinose inducible pBAD promoter. It will be appreciated by those of skill in the art that other promoters which can direct expression, preferably a high level of expression under appropriate fermentation conditions, would be effective as alternatives to the exemplified disclosures.
  • Nucleic acids and nucleic acid constructs may be constructed using any number of techniques standard in the art. For example, chemical synthesis, site directed mutagenesis, or recombinant techniques may be used. Such techniques are described, for example, in Sambrook et al (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ). Further exemplary techniques are described in the Examples section herein after. Essentially, the individual genes and regulatory elements will be operably linked to one another such that the genes can be expressed to form the desired proteins. Suitable vectors for use in the invention will be appreciated by those of ordinary skill in the art. However, by way of example, the following vectors may be suitable: pBAD or pMTL80000 vectors, and the plasmids exemplified in the Examples section herein after.
  • the invention also provides host organisms, particularly microorganisms, and including viruses, bacteria, and yeast, comprising any one or more of the nucleic acids described herein.
  • the invention also provides microorganisms which are capable of producing one or more of isopropanol, MEK, 2,3-butanediol and 2-butanol and optionally one or more other products, by fermentation of a substrate and which comprise at least one nucleic acid of the invention.
  • the microorganisms of the invention may be prepared from a parental microorganism using any number of techniques known in the art, including, for example, site directed mutagenesis techniques to introduce the desired mutation(s) into an alcohol dehydrogenase gene native to a parental microorganism, or other recombinant technologies to introduce one or more nucleic acid encoding one or more alcohol dehydrogeanse of the invention into a parental microorganism.
  • one or more exogenous nucleic acid encoding one or more alcohol dehydrogeanse is introduced into a parental microorganism and replaces one or more alcohol dehydrogenase gene native to the parental microorganism.
  • one or more exogenous nucleic acid encoding one or more alcohol dehydrogeanse of the invention is introduced to a parental microorganism and is supplementary to an alcohol dehydrogenase gene native to the parental microorganism.
  • one or more exogenous nucleic acid is introduced into a parental microorganism to introduce one or more desired mutation into one or more alcohol dehydrogenase gene native to the parental microorganism.
  • one or more exogenous nucleic acid encoding one or more alcohol dehydrogeanse is introduced into a parental microorganism, and one or more mutation is introduced to one or more alcohol dehydrogenase gene native to the parental microorganism to reduce or knock out its expression and activity.
  • a microorganism of the invention is prepared from a parental microorganism using recombinant technology.
  • a parental microorganism is transformed with one or more exogenous nucleic acid encoding an alcohol dehydrogeanse of the invention, or one or more nucleic acid adapted to introduce a desired mutation to a native alcohol dehydrogenase gene in the parental microorganism.
  • An exogenous nucleic acid may remain extra-chromosomal upon transformation of the parent microorganism or may integrate into the genome of the parent microorganism (in one embodiment to replace a native alcohol dehydrogenase gene, or introduce a mutation into a native alcohol dehyrogenase gene).
  • nucleotide sequences adapted to assist integration for example, a region which allows for homologous recombination and targeted integration into the host genome
  • extrachromosomal construct for example, origin of replication, promoter and other regulatory elements or sequences
  • transformation including transduction or transfection
  • transformation of a microorganism may be achieved by electroporation, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, or conjugation.
  • Suitable transformation techniques are described for example in, Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, 1989 .
  • One or more exogenous nucleic acids may be delivered to a parental microorganism as naked nucleic acids or may be formulated with one or more agents to facilitate the transformation process (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).
  • the one or more nucleic acids may be DNA, RNA, or combinations thereof, as is appropriate. Restriction inhibitors may be used; see, for example Murray, N.E. et al. (2000) Microbial. Molec. Biol. Rev. 64, 412 .)
  • the parental microorganism is a bacteria, Archae, and fungi.
  • the parental microorganism is chosen from genera Clostridium, Acetobacterium, Moorella, Butyribacterium, Blautia, Oxobacter, Thermoanaerobacter, Escherichia, Klebsiella, Zymomonas, Citrobacter, Enterobacter, Salmonella, Serratia, Lactobacillus, Lactococcus, Enterococcus, Pediococcus, Streptococcus, Saccharomyces, Pichia, Candida Hansenula, Yarrowia, Rhodotorula, Rhizopus, Trichosporon, Lipomyces, Aspergillus, trichoderma, Exophila, Mucor, Cladosporium, Phanerochaete, Cladiophilalophora, Paecilomyces, Scedosporium, Ophistoma, Bacillus, Oligotropha, Pseudomonas, Carbophilus, Hydrogenophaga
  • the parental microorganism is chosen from the group of carboxydotrophic acetogenic microorganisms, the group of ABE microorganisms, the group of Enterobacteria, the group of Lactobacillus, the group of fungi and yeasts, the group of aerobic carboxydotrophes, the group of aerobic CO 2 fixing organisms, the group of methylotrophes, and the group of methanogens.
  • the parental microorganism is selected from the group of carboxydotrophic acetogenic bacteria.
  • the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium coskatii, Clostridium aceticum, Clostridium magnum, Clostridium sp., Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.
  • carboxydotrophic acetogens are defined by their ability to utilize and grow chemoautotrophically on gaseous one-carbon (C1) sources such as carbon monoxide (CO) and carbon dioxide (CO2) with carbon monoxide (CO) and/or hydrogen (H2) as energy source under anaerobic conditions forming acetyl-CoA, acetate and other products.
  • C1 sources such as carbon monoxide (CO) and carbon dioxide (CO2) with carbon monoxide (CO) and/or hydrogen (H2) as energy source under anaerobic conditions forming acetyl-CoA, acetate and other products.
  • CODH Carbon monoxide dehydrogenase
  • Hydrogenase Formate dehydrogenase
  • Formyl-tetrahydrofolate synthetase Methylene-tetrahydrofolate dehydrogenase
  • Formyl-tetrahydrofolate cyclohydrolase Methylene-tetrahydrofolate reductase
  • Carbon monoxide dehydrogenase/Acetyl-CoA synthase CODH/ACS
  • the microorganism is selected from a cluster of carboxydotrophic Clostridia comprising the species C . autoethanogenum, C. ljungdahlii, and " C . ragsdalei” and related isolates. These include but are not limited to strains C . autoethanogenum JAI-1 T (DSM10061) (Abrini, N Weg, & Nyns, 1994), C . autoethanogenum LBS1560 (DSM19630) ( WO/2009/064200 ), C. autoethanogenum LBS1561 (DSM23693), C .
  • strains form a subcluster within the Clostridial rRNA cluster I (Collins et al., 1994), having at least 99% identity on 16S rRNA gene level, although being distinct species as determined by DNA-DNA reassociation and DNA fingerprinting experiments ( WO 2008/028055 , US patent 2011/0229947 ).
  • strains of this cluster are defined by common characteristics, having both a similar genotype and phenotype, and they all share the same mode of energy conservation and fermentative metabolism.
  • the strains of this cluster lack cytochromes and conserve energy via an Rnf complex.
  • strains of this cluster have a genome size of around 4.2 MBp (Köpke et al., 2010) and a GC composition of around 32 %mol (Abrini et al., 1994; Köpke et al., 2010; Tanner et al., 1993) ( WO 2008/028055 ; US patent 2011/0229947 ), and conserved essential key gene operons encoding for enzymes of Wood-Ljungdahl pathway (Carbon monoxide dehydrogenase, Formyl-tetrahydrofolate synthetase, Methylene-tetrahydrofolate dehydrogenase, Formyl-tetrahydrofolate cyclohydrolase, Methylene-tetrahydrofolate reductase, and Carbon monoxide dehydrogenase/Acetyl-CoA synthase), hydrogenase, formate dehydrogenase, Rnf complex ( rnfCDGEAB
  • strains all have a similar morphology and size (logarithmic growing cells are between 0.5-0.7 x 3-5 ⁇ m), are mesophilic (optimal growth temperature between 30-37 °C) and strictly anaerobe (Abrini et al., 1994; Tanner et al., 1993)( WO 2008/028055 ).
  • the traits described are therefore not specific to one organism like C. autoethanogenum or C . ljungdahlii , but rather general traits for carboxydotrophic, ethanol-synthesizing Clostridia. Thus, the invention can be anticipated to work across these strains, although there may be differences in performance.
  • the parental strain uses CO as its sole carbon and energy source.
  • the parental microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei. In one embodiment, the group also comprises Clostridium coskatii. In one particular disclosure, the microorganism is Clostridium autoethanogenum DSM10061 or DSM23693. In another particular disclosure, the microorganism is Clostridium ljungdahlii DSM13528 (or ATCC55383).
  • the parental microorganism is an ABE fermenting microorganism.
  • An "ABE fermenting microorganism” or “ABE microorganism” is a Gram-positive, Clostridial organism which is able to produce the solvents butanol, and ethanol, and acetone or isopropanol. Genera in this group include Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum. These organisms are all sporulating, Gram-positive and within the Clostridial rRNA cluster I. This group has been described in detail by Keis et al. (Keis, Shaheen, & Jones, 2001).
  • the ABE microorganism is selected from the group comprising Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum.
  • the parental microorganism is Clostridium acetobutylicum or Clostridium beijerinckii.
  • the microorganism is Clostridium acetobutylicum ATCC824 (DSM792) or EA 2018 (CCTCC M 94061).
  • the microorganism is Clostridium beijerinckii NCIMB8052 (ATCC51743) and NRRL B-593 (DSM 6423).
  • the parental microorganism is an Enterobacteria.
  • An Enterobacteria is a rod-shaped Gram-negative bacteria belonging to the order of Enterobacteriacea which is able to fermenting sugars to produce lactic acid, and/or ethanol, and/or acetoin, and/or 2,3-butabediol, and/or other products.
  • the Enterobacteria is selected from the group comprising Escherichia, Klebsiella, Zymomonas, Citrobacter, Enterobacter, Salmonella, Serratia.
  • the parental microorganism is Eschericia coli, Zymononas mobilis, Klebsiella pneumonia, Klebsiella oxtoca, Enterobacter cloacae or Serratia marcescens.
  • the parental microorganism is a Lactobacillus.
  • a Lactobacillus is a gram-positive lactic acid bacteria selected from the order of Lactobacillales which is able to fermenting sugars to produce lactic acid, and/or 2,3-butabediol, and/or MEK, and/or 2-butanol, and/or other products.
  • the Lactobacillus is selected from the group comprising Lactobacillus, Lactococcus, Enterococcus, Pediococcus, Streptococcus.
  • the parental microorganism is Lactobacillus brevis, Enterococcus faecalis, Lactococcus lactis.
  • the parental microorganism is a fungi or yeast.
  • Fungi are eukaryotic microorganisms
  • yeast are a specific subset thereof, which are able to ferment sugars to ethanol and/or acetoin, and/or other products.
  • the Fungi is selected from the group comprising Aspergillus, Trichoderma, Exophila, Mucor, Cladosporium, Phanerochaete, Cladiophilalophora, Paecilomyces, Scedosporium, Ophistoma.
  • the parental microorganism is Aspargillus niger or Trichderma resei.
  • the yeast is selected from the group comprising Saccharomyces, Pichia, Candida Hansenula, Yarrowia, Rhodotorula, Rhizopus, Trichosporon, Lipomyces and from the group comprising Aspergillus, Trichoderma, Exophila, Mucor, Cladosporium, Phanerochaete, Cladiophilalophora, Paecilomyces, Scedosporium, Ophistoma.
  • the parental microorganism is Saccharomyces cerevisiae, Candidia tropicalis, Candidia albicans or Yarrowia lipolytica.
  • the parental microorganism is Aspargillus niger or Trichderma resei.
  • parental the microorganism is an aerobic carboxydotroph.
  • Aerobic carboxydotrophes are bacteria that can be found ubiquitous in nature and have been isolated from various environments as well as humans (King and Weber, 2007). On taxonomic level, this physiological group is quite diverse, comprising of different phyla such as ⁇ -proteobacteria, firmicutes, or actinobacteria (King and Weber, 2007). All these organisms were shown to grown on CO levels > 1 % in presence of air (King and Weber, 2007).
  • a typical gas mix consists of 50 % CO and 50 % air (Cypionka et al., 1980).
  • the parental microorganism is selected from the group comprising Bacillus, Oligotropha, Pseudomonas, Carbophilus, Hydrogenophaga, Mycobacterium, Zavarzinia.
  • the parental microorganism is Oligotropha carboxydovorans, Carbophilus carboxidus, Hydrogenophaga pseudoflava, Mycobacterium sp., Pseudomonas carboxydohydrogena, Pseudomonas sp., Zavarzinia compransoris or Bacillus schlegelii.
  • the parental microorganism is an aerobic CO 2 fixing organism.
  • An aerobic CO2 fixing microorganism is an bacteria able to fix CO2 with H2 or via photosynthesis in present of oxygen.
  • the aerobic CO2 fixing microorganism is selected from the group comprising Cupravidus, Senechocystis, Chloroflexus.
  • the parental microorganism is Cupravidus necator, Senechocystis sp. or Chloroflexus auranticus.
  • the parental microorganism is a methylotroph.
  • Methylotrophic microrgansims are able to use reduced one-carbon substrates as methane or methanol as carbon source for growth.
  • the methylotrop is selected from the group comprising Methylomonas, Methylobacter, Methylococcus, Methylomicrobium, Methylosphera, Methylocaldum, Methylocystis, Methylosinus.
  • the parental microorganism is Methylococcus capsulatus or Methylosinus trichosporium.
  • the parental microorganism is a methanogen.
  • a methanogen is an Archeae that can reduce CO2 into methane.
  • the methanogen is selected from the group comprising Methanobacterium, Methanococcus, Methanogenium, Methanosarcina, Methanoshera, Methanothermobacter, Methanotrix.
  • the parental microorganism is Methanothermobacter marburgensis or Methanosarcina bakeri.
  • the invention provides a method for the production of isopropanol, ethanol, 2,3-butanediol and/or 2-butanol and optionally one or more other products by microbial fermentation of a substrate using a recombinant microorganism of the invention.
  • the invention provides a method for the production of one or more of acetoin, MEK, acetaldehyde and acetone, and optionally one or more other products.
  • the substrate is a substrate comprising one or more carbohydrate. In another embodiment, the substrate is a substrate comprising one or a combination of CO, CO 2 , and H 2 . In certain embodiments, mixed substrates comprising both one or more carbohydrate and a substrate comprising one or more of CO, CO 2 , and H 2 may be used.
  • the method comprises the steps of:
  • the one or more product includes isopropanol.
  • the method comprises the steps of:
  • the method may further comprise the step of recovering one or more products.
  • the one or more products are intermediates in the production of one or more downstream products.
  • the one or more products may be recovered and then used as a substrate in a separate fermentation or in a chemical synthesis reaction, for example.
  • the one or more products are not recovered and are converted to one or more downstream products in the same fermentation process.
  • a suitable liquid nutrient medium will need to be fed to the bioreactor.
  • the substrate and media may be fed to the bioreactor in a continuous, batch or batch fed fashion.
  • a nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used.
  • Suitable media for fermentation will be known in the art. However, by way of example, for fermentation of a substrate comprising one or more carbohydrate, Luria Broth (LB), Yeast Extract Peptone Dextrose (YEPD) or reinforced clostridia media (RCM) may be used.
  • Luria Broth Luria Broth
  • YEPD Yeast Extract Peptone Dextrose
  • RCM reinforced clostridia media
  • anaerobic media suitable for fermentation using CO are known in the art but by way of example, suitable media are described Biebel (2001). In one disclosure the media is as described in the Examples section herein after.
  • the fermentation should desirably be carried out under appropriate conditions for the substrate-to-the one or more product(s), and optionally one or more other product(s), fermentation to occur.
  • Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations to ensure that the substrate does not become limiting, and maximum product concentrations to avoid product inhibition.
  • reaction conditions will depend partly on the particular micro-organism of the invention used. However, in general, it is preferred that the fermentation be performed at pressure higher than ambient pressure. Also, since a given CO (and/or CO 2 and/or H 2 )-to-the one or more product(s) conversion rate is in part a function of the substrate retention time, and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment. According to examples given in US patent no. 5,593,886 , reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
  • WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively.
  • example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.
  • the rate of introduction of the CO (and/or CO 2 and/or H 2 )-containing gaseous substrate is such as to ensure that the concentration of CO (and/or CO 2 and/or H 2 ) in the liquid phase does not become limiting. This is because a consequence of CO (and/or CO 2 and/or H 2 )-limited conditions may be that the product(s) is consumed by the culture.
  • the composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction.
  • O 2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.
  • a culture of a microorganism of the invention is maintained in an aqueous culture medium.
  • the aqueous culture medium is a minimal anaerobic microbial growth medium.
  • Suitable media are known in the art and described for example in US patent no.s 5,173,429 and 5,593,886 and WO 02/08438 , and as described in the Examples section herein after.
  • the fermentation comprises the steps of anaerobically fermenting the substrate in a bioreactor to produce the one or more products using a recombinant microorganism of the invention.
  • Methods of this embodiment may be used to reduce the total atmospheric carbon emissions from an industrial process.
  • the method comprises the steps of:
  • the one or more products in step (b) above are acetoin, MEK, acetaldehyde and acetone, and optionally, one or more other products.
  • the method comprises the steps of:
  • the one or more products in step (b) above are acetoin, MEK, acetaldehyde and acetone, and optionally, one or more other products.
  • the gaseous substrate fermented by the microorganism is a gaseous substrate containing CO.
  • the gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
  • the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.
  • the CO may be a component of syngas (gas comprising carbon monoxide and hydrogen).
  • syngas gas comprising carbon monoxide and hydrogen.
  • the CO produced from industrial processes is normally flared off to produce CO 2 and therefore the invention has particular utility in reducing CO 2 greenhouse gas emissions and producing butanol for use as a biofuel.
  • the gaseous substrate may be filtered or scrubbed using known methods.
  • Isopropanol, 2,3-butanediol, ethanol, 2-butanol, acetoin, MEK, acetaldehyde and/or acetone or a mixed stream containing any one or more of these products and acetone and optionally one or more other products may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, gas stripping and extractive fermentation, including for example, liquid-liquid extraction.
  • the one or more products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more products from the broth.
  • Alcohols may conveniently be recovered for example by distillation.
  • Acetone may be recovered for example by distillation.
  • Any acids produced may be recovered for example by adsorption on activated charcoal.
  • the separated microbial cells are preferably returned to the fermentation bioreactor.
  • the cell free permeate remaining after any alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.
  • the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • Clostridium including C . ljungdahlii (Köpke, et al ., 2010) have been shown to utilise CO as a sole carbon source, with ethanol as the end product.
  • the bacteria are able to fix CO and convert it to acetyl-CoA via the Wood-Ljungdahl pathway.
  • the acetyl moiety of the acetyl CoA can then be used in a variety of metabolic pathways.
  • the carbonyl group can be reduced to its corresponding alcohol by an alcohol dehydrogenase (ADH) enzyme (Köpke, et al ., 2010). This provides a pathway to convert CO into commercially valuable biofuels and biochemicals.
  • ADH alcohol dehydrogenase
  • Genome sequencing of a strain of C . autoethanogenum DSM10061 identified an ADH that is 86% identical to the previously-characterized enzyme from C . beijerinckii.
  • the ADH from this strain is able to utilise acetaldehyde as its substrate and produce ethanol as the end product. It is also able to catalyse the reduction of acetone to isopropanol.
  • Isopropanol is a more economically valuable final product than ethanol, as it can be dehydrated to form propylene, which can be polymerised to polypropylene, a commonly used plastic (Inokuma et al., 2010).
  • a microbial route to propylene will also decrease the demand for petroleum, from which most propylene is currently derived.
  • E. coli DH5 ⁇ -E was obtained from Invitrogen.
  • the genotype of this strain is: F-80 ⁇ lacZM15 (lacZYA-argF) U169 recA1 endA1 hsdR17(rk-, mk+) gal- phoA supE44 - thi-1 gyrA96 relA1.
  • E. coli LMG194 was obtained from Invitrogen. The genotype of this strain is: F- ⁇ lacX74 galE thi rpsL ⁇ phoA (Pvu II) ⁇ ara714 leu::Tn10.
  • E. coli MC1061 was obtained from Coli Genetic Stock Centre. The genotype of this strain is araD139 ⁇ (araA-leu)7697 ⁇ (lac)X74 galK16 galE15(GalS) lambda- e14- mcrA0 relA1 rpsL150(strR) spoT1 mcrB1 hsdR2.
  • Clostridium autoethanogenum DSM10061 was obtained from DSMZ (The German Collection of Microorganisms and Cell Cultures, Inhoffen No 7 B, 38124 Braunschweig, Germany).
  • E. coli strains were cultivated in aerobic conditions, using Luria Burtani medium supplmented with either ampicillin (100 ⁇ g/mL) or carbenicillin (50 ⁇ g/mL). [098] Solid media contained 1.5% agar. All strains were grown at 37°C unless otherwise noted.
  • SOC medium (20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, 2.5 mM KCl and 20 mM glucose) was used for recovery of E. coli after electroporation.
  • Clostridium autoethanogenum was grown in PETC media with pH5.6 (Tab. 1) and C. acetobutylicum and C . beijerinckii in RCM media (Tab. 2) using standard anaerobic techniques [ Hungate RE: A roll tube method for cultivation of strict anaerobes, in Norris JR and Ribbons DW (eds.), Methods in Microbiology, vol. 3B. Academic Press, New York, 1969: 117-132 ; Wolfe RS: Microbial formation of methane. Adv Microb Physiol 1971, 6: 107-146 ].
  • PETC medium Media component Concentration per 1.0L of media NH 4 Cl 1g KCl 0.1 g MgSO 4 .7H 2 O 0.2 g NaCl 0.8 g KH 2 PO 4 0.1 g CaCl 2 0.02 g Trace metal solution (see below) 10 ml Wolfe's vitamin solution (see below) 10 ml Yeast Extract (optional) 1 g Resazurin (2 g/L stock) 0.5 ml NaHCO 3 2 g Reducing agent 0.006-0.008 % (v/v) Fructose (for heterotrophic growth) 5 g Trace metal solution per L of stock Nitrilotriacetic Acid 2 g MnSO 4 .H 2 O 1 g Fe (SO 4 ) 2 (NH 4 ) 2 .6H 2 O 0.8 g CoCl 2 .6H 2 O 0.2 g ZnSO 4 .7H 2 O 0.2 mg CuCl 2 .2H 2 O 0.02 g Na
  • amino acid and nucleic acid sequence of the wild-type ADH of C . autoethanogenum DSM10061 are shown in SEQ ID 36 and SEQ ID 35, respectively.
  • pMTL85147-ThlA-CtfAB-Adc-Adh ( Fig. 8 ) was used for amplification of C . autoethanogenum DSM10061 ADH gene.
  • Plasmid pMTL85147-ThlA-CtfAB-Adc-Adh has been constructed usingstandard Recombinant DNA and molecular cloning techniques [ Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989 ; Ausubel FM, Brent R, guitarist RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current protocols in molecular biology. John Wiley & Sons, Ltd., Hoboken, 1987 ]. ThlA gene (NC_003030.1; GI: 1119056) was amplified from genomic DNA of C .
  • genes adc-ctfAB-adc (NC_009617; region: 4,400,524-4,402,656; including GI: 5294994, GI: 5294995, and GI: 5294996) were amplified from C . beijerinckii , and adh gene ( Fig. 5 ) and Wood-Ljungdahl promoter region (SEQ ID 39) were amplified from C . autoethanogenum DSM10061. Oligonucleotide sequences used for amplification are given in Tab. 4.
  • Genomic DNA from Clostridium acetobutylicum ATCC824, C . beijerinckii NCIMB8052 and C . autoethanogenum DSM10061 was isolated using a modified method by Bertram and Dürre (Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch Microbiol 1989, 151: 551-557 ).
  • a 100-ml overnight culture was harvested (6,000 x g, 15 min, 4 °C), washed with potassium phosphate buffer (10 mM, pH 7.5) and suspended in 1.9 ml STE buffer (50 mM Tris-HCl, 1 mM EDTA, 200 mM sucrose; pH 8.0).
  • the expression vector pBAD(KpnI)-WpiMetC was obtained from Invitrogen.
  • the plasmid used in this study had been modified previously, by the insertion of a fragment encoding a hexahistidine (His 6 ) tag, a TEV protease cleavage site (for removing His 6 from ADH, if desired, after purification), and the gene encoding the MetC enzyme from an unrelated bacterium ( Wolbachia pipientis ).
  • the wild type ADH C . autoethanogenum DSM10061 was amplified from a 50ng/ ⁇ l working stock of the plasmid pMTL85147-ThlA-CtfAB-Adc-Adh(LZ) using the primers ADH_TEV_KpnI_for and ADH_HindIII_Reverse.
  • a 50 ⁇ L PCR mixture was made as follows: Reagent Initial Conc Final Conc Vol/reaction Phusion Buffer 5x 1x 10 ⁇ L dNTPs 2mM 200 ⁇ M each dNTP 5 ⁇ L ADH_TEV_KpnI_for 10mM 0.5 ⁇ M 2.5 ⁇ L ADH_HindIII_Rev 10Mm 0.5 ⁇ M 2.5 ⁇ L Phusion Polymerase 2U/ ⁇ L 1 U/reaction 0.5 ⁇ L pMTL85147-ThlA- CtfAB-Adc-Adh(LZ) template DNA 50ng/ ⁇ L 20ng 0.4 ⁇ L Water 29.1
  • the pBAD backbone was prepared by digesting pBAD(KpnI)-WpiMetC with the enzymes KpnI-HF and HindIII, with the following recipe: Reagent Initial Concentration Final Concertration Volume in Reaction NEB Buffer 4 10X 1X 5 ⁇ L pBAD(KpnI)-WpiMetC 140ng/ ⁇ L 4.1 ⁇ g 28 ⁇ L KpnI-HF 20,000U/mL 10U/reaction 0.5 ⁇ L HindIII 20,000U/mL 10U/reaction 0.5 ⁇ L Water 16 ⁇ L
  • the amplified ADH gene was similarly digested with KpnI-HF and HindIII: Reagent Initial Concentration Final Concentration Volume in Reaction NEB Buffer 4 10X 1X 5 ⁇ L WT ADH 75ng/ ⁇ L 1.8 ⁇ g 25 ⁇ L KpnI-HF 20,000U/mL 10U/reaction 0.5 ⁇ L HindIII 20,000U/mL 10U/reaction 0.5 ⁇ L Water 19 ⁇ L
  • the reaction was incubated at 16°C for 2 hours.
  • a 2 ⁇ L aliquot of the ligation mixture was used to transform a 50 ⁇ L aliquot of E. coli MC1061 cells by electroporation. After an hour recovery at 37°C in SOC, aliquots were spread on LB-ampicillin plates and incubated at 37°C overnight. 6 colonies were picked and screened for the presence of the ADH insert by PCR using the pBAD_for and ADH_HindIII_rev primers.
  • a freezer stock was made of a successful clone, with 700 ⁇ l culture and 300 ⁇ l sterile 50% (v/v) glycerol. The sequence of the ADH gene was verified by DNA sequencing.
  • the first step in site directed mutagenesis was the generation of two overlapping primary products.
  • the PCR reactions for these are as follows:
  • the next step in site directed mutagenesis was to use overlap extension PCR ( Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77, 51-59 ) with the outside primers, to recombine the two primary products into a full-length secondary product that contained the complete gene with the introduced mutation. The two primary products are mixed so that they are in equal molar concentrations.
  • a 5mL LB-ampicillin overnight culture from freezer stocks was grown at 37°C, and used to inoculate 100mL LB-ampicillin the following morning.
  • the culture was incubated at 37°C until an OD 600 of 0.8 was reached.
  • 1mL of 20% arabinose was added and the culture was incubated at 28°C for the remainder of the expression.
  • Pellets were then resuspended in HEPES buffer (50 mM Na-HEPES and 0.2 mM DTT, pH 8.0), and 0.2 ⁇ L each of Benzonase (Merck, 25 units/ ⁇ L) and rLysozyme (Merck, 30 kU/ ⁇ L) were added. The mixtures were incubated at room temperature for 15min and then frozen (at -80°C) and thawed 3 times. 10 ⁇ L was taken at this point from each mixture as 'total protein' samples. The remainder of the sample was centrifuged at 13000rpm for 1min, and the supernatant removed and placed on ice.
  • HEPES buffer 50 mM Na-HEPES and 0.2 mM DTT, pH 8.0
  • Benzonase Merck, 25 units/ ⁇ L
  • rLysozyme Merck, 30 kU/ ⁇ L
  • the frozen cell pellets described above(from 100mL cultures) were resuspended in 10mL ice-coldlysis buffer (50 mM potassium phosphate, 300 mM NaCl, pH 7.0), with 0.5 ⁇ l rLysozyme (30kU/ ⁇ l), 0.5 ⁇ l Benzonase(25U/ ⁇ l), and 50 ⁇ l protease inhibitor cocktail (Sigma).
  • the cells were lysed by sonication, insoluble debris was pelleted, and the supernatant was clarified using a 0.2 micron filter.
  • the clarified supernatant was added to Talon resin (Clontech) which had been thoroughly washed with lysis buffer.
  • a bed volume of 500 ⁇ L was used for purifying the wild-type ADH, and a bed volume of 200 ⁇ L was used for purifying each mutant.
  • the protein was allowed to bind to the Talon resin for an hour at 4°C and then washed several times with lysis buffer.
  • the protein was eluted from the column using lysis buffer supplemented with 150mM imidazole, and the eluant was collected in 500 ⁇ L fractions.
  • the wild-type protein was highly soluble when expressed in E. coli, and could be readily purified ( Figure 1 ).
  • the Ser199Asp and Arg200Gln mutant proteins were also highly soluble ( Figure 2 ).
  • the solubility of the other two point mutants (Ser199Glu and Arg200Glu) were more variable (results not shown).
  • the double mutant Ser199Glu + Arg200Gln was more soluble than Ser199Glu (results not shown).
  • sequence of the Ser199Asp mutant is provided in SEQ ID 37 (nucleic acid) and SEQ ID 38 (amino acid).
  • assays had a cofactor (NADPH/NADH) concentration of 0.2 mM, a substrate (acetaldehyde, acetyl-CoA, acetone, DL-acetoin, MEK) concentration of 3 mM and an ADH concentration of 30 nM. They were carried out in 50 mM Tris-HCl buffer (pH 7.5), with 1mM DTT. All assays were done in triplicate, with freshly prepared substrates and cofactors.
  • kinetics for the purified wild-type enzyme ( Fig. 12 ) of C . autoethanogenum DSM10061 were determined as baseline to evaluate mutated enzymes with substitutions ( Fig. 9 ).
  • Activity could be detected with ketones (acetone, MEK, acetoin) and aldehydes (acetaldehyde) which are important in several fermentation pathways ( Fig. 14 ).
  • the Ser199Asp mutant was nearly as active as wild-type with acetone ( Figure 3 ). Most interestingly, the Ser199Asp mutant is more specific for acetone ( Figure 4 ). This indicates that replacing wild-type ADH with the Ser199Asp mutant may lead to increased isopropanol production. This mutant was the basis for further substitutions as described in Example 2.
  • Example 1 Based on the results from Example 1, the inventors generated and studied the activity of a further 8 alcohol dehydrogenase mutants with 1 to 4 amino acid substitutions: Name Mutation(s) Mutant 2 S199E Mutant 7 G198D, S199V, P201E Mutant 8 G198E, S 199V, P201E Mutant 9 G198D, S199L, P201E Mutant 10 G198D, S199V, P201E, Y218F Mutant 11 G198D, S199V, P201E, Y218A Mutant 12 G198D, S199D, P201E Mutant 13 Y218A
  • the gene sequence for Mutant 7 was synthesized by DNA 2.0. The sequence was codon optimized for expression of the protein in E. coli. The synthesized gene was provided in the plasmid pJ201. This sequence included HindIII and KpnI restriction sites for subcloning the Mutant 7 ADH gene into the expression vector pBAD(KpnI)-ADH.
  • the pBAD backbone was prepared by digesting pBAD(KpnI)-ADH with the restriction enzymes KpnI-HF and HindIII-HF.
  • the Mutant 7 gene was similarly digested from the pJ201 vector with the restriction enzymes Kpn-HF and HindIII-HF.
  • the digested products were separated on a 1% agarose gel stained with SYBRsafe (Invitrogen). The bands corresponding to the digested vector and insert were excised and the DNA was recovered using a gel clean-up kit (Omega Bio-Tek).
  • the purified insert and vector DNA were ligated using a 3:1 molar ratio of insert to vector, using T4 DNA ligase (NEB) according to the manufacturer's standard protocol.
  • NEB T4 DNA ligase
  • the ligation mixture was used to transform E. coli MC1061 cells by electroporation. Aliquots of the transformed cells were spread on LB-ampicillin and incubated at 37°C overnight. A single colony was picked. The resulting expression plasmid was purified, and the sequence of the Mutant 7 gene was confirmed by DNA sequencing. A freezer stock was made of the successful clone.
  • the template DNA used for the construction of Mutants 8, 9, 10, 11 and 12 was pBAD(KpnI)-Mutant 7 ADH.
  • the template DNA used for the construction of Mutant 13 was pBAD(KpnI)-ADH.
  • the protocol for constructing Mutants 8-13 was the same.
  • Each mutant was constructed using the Quikchange II Site-Directed Mutagenesis Kit from Stratagene, using their standard recommended protocol. The forward and reverse primers used for the construction of each mutant are listed in Table 5.
  • the products of the Quikchange mutagenesis reaction were used to transform chemically-competent E . coli XL1-Blue cells by heat shock. Single colonies were picked. The resulting expression plasmids were purified, and the sequence of each mutant gene was confirmed by DNA sequencing. A freezer stock was made of each successful clone.
  • the expression vectors for Mutants 2, 7, 10, 11 were used to transform E. coli LMG194 that had previously been transformed with plasmid pGro7 (Takara Bio, Inc.). This plasmid facilitates arabinose-inducible expression of the GroEL/ES chaperone proteins.
  • the expression vector for Mutant 13 was used to transform E. coli LMG194.
  • each ADH mutant was induced in mid-log phase cultures (OD 600 ⁇ 0.5), by adding L-arabinose to a final concentration of 0.2% (w/v). The cultures were incubated at 28°C for an additional 5 h. Cells were harvested by centrifugation and the pellets were stored at -80°C. Each pellet was resuspended in 10 mL of lysis buffer (50 mM potassium phosphate, 300 mM NaCl, pH 7.0). Protease inhibitor cocktail (150 ⁇ L), Benzonase nuclease (37.5 U) and lysozyme (0.2 mg.mL -1 , final concentration) were added.
  • lysis buffer 50 mM potassium phosphate, 300 mM NaCl, pH 7.0
  • Protease inhibitor cocktail 150 ⁇ L
  • Benzonase nuclease 37.5 U
  • lysozyme 0.2 mg.mL -1 , final concentration
  • each purified protein was eluted with 5 bed volumes of elution buffer (50 mM potassium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7.0).
  • Amicon Ultra centrifugal filter units (10 kDa molecular weight cut-off; Merck Millipore, Billerica, MA) were used to exchange the purified protein into storage buffer (50 mM potassium phosphate, 150 mM NaCl, 10% (v/v) glycerol, pH 7.5).
  • Mutants 2, 7 and 11 were all highly soluble when expressed in E. coli with the pGro7 plasmid. The yield soluble protein for Mutant 10 was lower than the other variants. Mutants 8, 9 and 12 were completely insoluble, even when co-expressed with pGro7. Mutant 13 was highly soluble, and could be produced in high quantities under the same conditions as the wild-type ADH protein.
  • Mutant 11 has the highest activity using NADH as the cofactor ( Figure 6 ).
  • the cofactor usage for Mutant 11 has completely switched (compared to wild-type ADH); Mutant 11 had no detectable activity with NADPH.
  • Mutant 11 has four mutations (G198D, S199V, P201E, Y218A). All four mutations are required for the observed switch in cofactor usage. The addition of the Y218A mutation was required for activity with NADH, but this mutation alone (i.e. Mutant 13) had no effect on cofactor preference. This could have important advantages in fermentation pathways to Isopropnaol, 2,3-butanediol, 2-butanol, and ethanol as shown in Fig. 14
  • mutants are also more specific for acetone, over either a larger substrate (acetoin and MEK) or a smaller substrate (acetaldehyde). All mutants (2, 7, 10 and 11) have an increased substrate specificity of acetone over MEK. Mutants 2, 10 and 11 have an increased substrate specificity of acetone over acetaldehyde and over acetoin.
  • Mutant 2 10 and 11 have an increased substrate specificity of MEK over acetaldehyde and over acetoin.
  • mutants are also more specific for acetaldehyde over a larger substrate (acetone, acetoin and MEK).
  • Mutant 7 has an increased substrate specificity of acetaldehyde over acetone and over MEK.
  • Mutants 2, 7 and 10 have an increased substrate specificity of acetaldehyde over acetoin.
  • mutants are also more specific for acetoin over a smaller substrate (acetone, acetoin and MEK).
  • Mutant 7 has an increased substrate specificity of acetoin over acetone and over MEK.
  • Mutants 7 and 11 have an increased substrate specificity of acetoin over acetaldehyde.
  • Mutant 10 lost the activity with acetoin, while still having activity with the other substrates acetone, MEK and acetaldehyde.
  • D-acetoin or (S)-acetoin:: D-Acetoin was synthesized as described via the regioselectively-controlled monooxidation of D-(-)-2,3-butanediol ( D'Accoloti et al. 1993 J. Org. Chem. 58: 3600-1 ) The requisite dimethyldioxirane (DMDO)-acetone solution was freshly prepared according to a modification of a previously reported procedure and subsequently titrated by iodometry.
  • DMDO dimethyldioxirane
  • DMDO-acetone solution A 1000 mL 3-neck round bottom flask was fitted with a gas adaptor and a double distillation head, to which was attached a vacuum adapted receiver. The final joint was stoppered and intended for reagent additions. A round-bottom flask was fastened to the receiving end and cooled to -78 °C in an acetone-dry ice bath. In line with the vacuum was a Buchi® cold finger condenser cooled to -20 °C using salted-ice, followed by connection to a -196 °C liquid nitrogen cold finger trap.
  • the reaction vessel was charged with a large oval stirring bar, water (220 mL), acetone (160 mL) and sodium bicarbonate (120.063 g). Vigorous stirring was initiated at 0 °C whilst purging with nitrogen, which was continued during addition of solid oxone (250.047 g) in 5 portions at 3 minute intervals. Strong effervescence was exhibited and the rate of oxone addition was controlled to manage this accordingly. After addition of the second portion of oxone, the colourless solution above the white slurry took on a pink colour. Stirring was continued for 15 minutes upon complete addition of oxone.
  • Carboxydotropic organism C. autoethanogenum was shown to produce both ethanol and 2,3-butanediol from CO (Köpke et al., 2011) ( Fig. 14 ).
  • an alcohol dehydrogenase is present in C. autoethanogenum that catalyses the last step in both the ethanol and the 2,3-butanediol pathway, the reactions of acetaldehyde to ethanol respectively acetoin to 2,3-butanediol ( Fig. 14 ).
  • the activity of this wild-type enzyme with acetaldehyde is higher than with acetoin ( Fig.
  • an alcohol dehydrogenase mutant (mutant 7) was generated with amino acid substitutions G198E, S199V, P201E that has an improved substrate specificity for acetoin over acetaldehyde ( Fig. 7 ).
  • This enzyme that preferentially uses acetoin over acetaldehyde compared to the wild-type enzyme can be used to increase the 2,3-butanediol production over ethanol production compared to the wild-type strain.
  • the gene (Seq. ID 41) of a mutant alcohol dehydrogenase is cloned into a pMTL85353 shuttle vector (Heap, Pennington, Cartman, & Minton, 2009) carrying a ferredoxin promoter using sites NdeI and EcoRI.
  • the construct is then methylated and transformed into C. autoethanogenum by electroporation as described ( US 2012/0252083 , WO/2012/115527 ). Thiamphenicol resistant colonies are picked and grown in 5 mL liquid media.
  • the transformed culture is verified by PCR and a fermentation experiment is carried out.
  • the strain carrying the mutant alcohol dehydrogenase will have an increased 2,3-butanediol:ethanol ratio.
  • C . ljungdahlii can be modified with the alcohol dehydrogenase mutant, using the same plasmid. Electroporation has been described for several carboxydotrophic acetogens such as C . ljungdahlii ( Köpke et al. 2010, Poc. Nat. Acad. Sci. U.S.A. 107: 13087-92 ; ( Leang, Ueki, & Lovley, 2011) PCT/NZ2011/000203 ; WO2012/053905 ), Acetobacterium woodii ( Straetz et al., 1994, Appl. Environ. Microbiol.
  • Example 4 Preferential production of ethanol over 2,3-butanediol in carboxydotrophes using optimized alcohol dehydrogenase
  • C . autoethanogenum is able to produce both ethanol and 2,3-butanediol from CO ( Fig. 14 ).
  • this can be achieved by inactivating an enzyme in the pathway of one of the pathways.
  • this strategy can't be applied, which is the case for the alcohol dehydrogenase of C . autoethanogenum that catalyzes both the reduction of acetaldehyde to ethanol and acetoin to 2,3-butanediol.
  • the current invention gives an alternative means to achieve, for example, production of ethanol with reduced levels or without 2,3-butanediol.
  • an alcohol dehydrogenase mutant (mutant 10) was generated with amino acid substitutions G198D, S199V, P201E, Y218F that lost the ability to reduce acetoin, but still has activity with acetaldehyde ( Fig. 7 ).
  • the gene (Seq. ID 47) of this mutant alcohol dehydrogenase is cloned into a vector along with the flanking regions of the alcohol dehydrogenase to allow a double homologous crossover integration (replacing the wild-type with the mutant alcohol dehydrogense). 1 kb 5' (Seq. ID.
  • the two PCR products are cloned into pMTL85151 plasmids between the SbfI/ NotI and NheI/AscI sites to get pMTL85151-SecAdh-KO.
  • the vector is transformed as described above. Following selection on thiamphenicol plates the transformants are screened for swap out of the wild-type alcohol dehydrogenase with the mutant alcohol dehydrogenase using the primers SecOf (TTGGAATTTTAGCTGTAGATAACAA (SEQ ID 61)) and SecOr (TAAGTGATTTTCAATGGACTTTACT (SEQ ID 62)) that flank the homology arms.
  • C. autoethanogenum has been modified for isopropanol production from CO by introducing acetone biosynthesis genes, relying on the wild-type alcohol dehydrogenase ( US 2012/0252083 , WO/2012/115527 ).
  • a highly acetone specific mutant alcohol dehydrogenase (mutant 11) with substitutions G198D, S199V, P201E, Y218A generated in example 2 can be introduced.
  • the mutant alcohol dehydrogenase gene (Seq. ID 53) is cloned into acetone biosynthesis plasmid (Seq. ID 40) by SalI/XhoI restriction sites. The plasmid is then transformed in C . autoethanogenum as described above.
  • Fermentation experiments with the transformed culture will show an increased isopropanol production in which all acetone is converted to isopropanol.
  • the organism will use NADPH and NADH for isopropanol synthesis, allowing to tap into both pools.
  • C . ljungdahlii can be modified with the alcohol dehydrogenase mutant, using the same plasmid. Electroporation has been described for several carboxydotrophic acetogens as C . ljungdahlii ( Köpke et al. 2010, Poc. Nat. Acad. Sci. U.S.A. 107: 13087-92 ; ( Leang et al., 2011) PCT/NZ2011/000203 ; WO2012/053905 ), Acetobacterium woodii ( Straetz et al., 1994, Appl. Environ. Microbiol.
  • C . acetobutylicum can be metabolically engineered for isopropanol production using a secondary alcohol dehydrogenase from C . beijerinckii, but the reported results show only low isopropanol levels with residual acetone that hasen't been converted through to isopropanol [ Lee et al, 2012: Metabolic engineering of Clostridium acetobutylicum ATCC824 for isopropanol-butanol-ethanol fermentation, Appl. Environ. Microbiol. 78: 1416-1423 ]. To improve this process an optimized alcohol dehydrogenase is required to overcome this limitation.
  • an optimized mutant alcohol dehydrogenase (mutant 11) was generated with substitutions G198D, S199V, P201E, Y218A that has high specificity towards acetoin and is also able to use NADH, which is more abundant than NADPH in C . acetobutylicum.
  • the mutant alcohol dehydrogenase gene (Seq. ID 53) is cloned into into a pMTL85354 shuttle vector (Heap et al., 2009) carrying the strong C. acetobutylicum thiolase promoter using sites NdeI and EcoRI.
  • the plasmid is then in vivo methylated using a Bacillus subtilis phage methyltrasnferase and transformed in C. acetobutylicum as described (Mermelstein & Papoutsakis, 1993). In a fermentation carried out with the transformed culture all acetoin is converted to isopropanol with high specificity.
  • E. coli has been a target for isopropanol production in several studies [ Hanai T et al (2007) Engineered synthetic pathway for isopropanol production in Escherichia coli. Applied and environmental microbiology 73:7814-8 ; Inokuma K et al (2010) Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping. Journal of bioscience and bioengineering 110:696-701 ; Jojima T et al (2008) Production of isopropanol by metabolically engineered Escherichia coli. Applied microbiology and biotechnology 77:1219-24 ]. However all studies use the very same non-optimized alcohol dehydrogenase from C .
  • an optimized mutant alcohol dehydrogenase (mutant 11) was generated with substitutions G198D, S199V, P201E, Y218A that has high specificity towards acetoin and is also able to use NADH.
  • E.coli can be engineered to include this mutant alcohol dehydrogenase using standard techniques used in the art. The recombinant E.coli will have increased isopropanol production compared to a wild type organism.
  • D-Acetoin (or (S)-Acetoin) can be converted to meso-2,3-butanediol by action of the alcohol dehydrogenase of C . autoethanogenum described in example 1. Conversion of meso -2,3-Butanediol to MEK has been described with diol dehydratase enzyme of for example A.
  • Codon optimized gene for Mutant 11 (Seq. ID 50) and codon optimized genes for diol dehydratase from Klebsiella pneumonia (YP_002236782; YP_002236783; YP_002236784) are cloned under inducible yeast promoter GAL1/10 into an appropriate vector as described (Steen et al., 2008). Transformation of all S. cerevisiae strains is performed using the lithium acetate method as described ( Gietz RW: RA Guide to Yeast Genetics and Molecular and Cell Biology. Part B. San Diego, CA: Academic Press Inc; 2002:87-96 ). After successful transformation and verification a fermentation with S. cerevisiae in rich YPD medium at 30°C is carried out with 2-butanol and low levels of ethanol as product.

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Claims (14)

  1. Alkoholdehydrogenase, die aus SEQ ID NO: 42, SEQ ID NO: 48 oder SEQ ID NO: 50 ausgewählt wird.
  2. Nukleinsäure, die eine Alkoholdehydrogenase nach Anspruch 1 kodiert.
  3. Nukleinsäure nach Anspruch 2, wobei die Nukleinsäure SEQ ID NO: 41, SEQ ID NO: 47 oder SEQ ID NO: 49 umfasst.
  4. Nukleinsäurevektor, umfassend die Nukleinsäure nach Anspruch 2.
  5. Rekombinanter Mikroorganismus, umfassend eine Nukleinsäure nach Anspruch 3.
  6. Rekombinanter Mikroorganismus nach Anspruch 5, wobei der rekombinante Mikroorganismus in der Lage ist, eines oder mehreres von Isopropanol, Ethanol, 2,3-Butandiol, 2-Butanol, Acetoin, MEK, Acetaldehyd und Azeton und optional ein oder mehrere andere Produkte zu produzieren.
  7. Rekombinanter Mikroorganismus nach Anspruch 5, wobei der rekombinante Mikroorganismus aus der Gruppe bestehend aus Bakterien, Archaeen und Pilzen ausgewählt ist.
  8. Rekombinanter Mikroorganismus nach Anspruch 5, wobei der rekombinante Mikroorganismus ein carboxytrophes Acetogen ist, das aus der Gruppe bestehend aus Acetobacterium woodii, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Moorella thermoacetica, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium coskatii, Clostridium aceticum, Clostridium magnum, Clostridium sp., Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, MooreIla thermoacetica, MooreIla thermautotrophica, Oxobacter pfennigii und Thermoanaerobacter kiuvi ausgewählt ist.
  9. Rekombinanter Mikroorganismus nach Anspruch 5, wobei der rekombinante Mikroorganismus ein Mikroorganismus zur ABE-Fermentation ist, der aus der Gruppe bestehend aus Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum und Clostridium saccharoperbutylacetonicum ausgewählt ist.
  10. Rekombinanter Mikroorganismus nach Anspruch 5, wobei der rekombinante Mikroorganismus aus der Gruppe umfassend Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Bacillus licheniformis, Bacillus subtilis, Lactobacillus brevis und Saccharomyces cerevisiae ausgewählt ist.
  11. Verfahren zur Produktion von einem oder mehreren von Isopropanol, Ethanol, 2,3-Butandiol, 2-Butanol, Acetoin, MEK, Acetaldehyd und Azeton und optional von einem oder mehreren anderen Produkten durch mikrobielle Fermentation eines Substrats unter Verwendung eines Mikroorganismus nach einem der Ansprüche 5 bis 10.
  12. Verfahren nach Anspruch 11, wobei das Substrat aus einem Substrat, umfassend eines oder mehreres von CO, CO2 und H2, und/oder einem Substrat, umfassend ein oder mehrere Kohlenhydrate, ausgewählt ist.
  13. Verfahren nach Anspruch 11, wobei das Substrat ein durch einen industriellen Prozess produziertes CO-haltiges Gas umfasst.
  14. Verfahren nach Anspruch 13, wobei der industrielle Prozess aus der Gruppe bestehend aus Eisenmetallproduktherstellung, Nichteisenproduktherstellung, Erdölraffinationsprozessen, Kohlevergasung, Biomassenvergasung, Stromerzeugung, Rußerzeugung und Koksherstellung ausgewählt ist.
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JP5923679B1 (ja) * 2015-01-26 2016-05-25 有限会社情報科学研究所 還元発酵方法、還元発酵装置、酸化還元発酵方法、及び酸化還元発酵装置
BR112017018257A2 (pt) 2015-02-27 2018-04-10 White Dog Labs Inc método de fermentação mixotrópica para produzir acetona, isopropanol, ácido butírico e outros bioprodutos, e misturas dos mesmos
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EP2834351A1 (de) 2015-02-11
NZ700609A (en) 2016-07-29
US20130267006A1 (en) 2013-10-10
EP2834351A4 (de) 2016-01-13
JP2015512646A (ja) 2015-04-30
KR102079274B1 (ko) 2020-02-20
WO2013152236A1 (en) 2013-10-10
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US9550979B2 (en) 2017-01-24
CN104619834B (zh) 2018-06-15

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